Method for analyzing body fluids for the presence of cancer cells, use thereof, corresponding analysis kits, and use of specific active substances for treating cancer

ABSTRACT

The present invention relates to a method for investigating body fluids for cancer cells, the use thereof and corresponding analysis kits, and the possibilities for cancer treatment derived therefrom. The method is based essentially on determining the expression of the manganese superoxide dismutase, thioredoxin reductase and/or glutathione peroxidase genes. Use of this method permits in particular reliable tumor diagnosis and prognosis. Diminishing an elevated expression of these genes has therapeutic value and may be utilized for cancer treatment.

The present invention relates to a method for investigating body fluidsfor cancer cells, the use thereof and corresponding analysis kits, andthe possibilities for cancer treatment derived therefrom. The method isbased essentially on determining the expression of the manganesesuperoxide dismutase, thioredoxin reductase and/or glutathioneperoxidase genes. The use of this method permits in particular reliabletumor diagnosis and prognosis. Diminishing an elevated expression ofthese genes may have therapeutic value and be utilized for cancertreatment.

Aerobic organisms in particular are exposed to oxidative stressthroughout life. Both endogenous and exogenous factors lead tocontinuous production of free radicals, especially in the form ofreactive oxygen species. Without an appropriate antioxidativeprotection, the damage, associated with the reaction of the freeradicals, to cellular constituents and cellular structures would soonresult in death of the cell.

Although the organism is able to avoid most of the oxidative damage, theantioxidative protection, which is very complex and consists of severalhundred components in each individual cell, does not appear to becomprehensive. Instead, it must be assumed that oxidative damageaccumulates with increasing age, suggesting that this is an essential,if not the decisive, factor in the aging process. The development ofcancer is also discussed in this connection.

There are at least three different superoxide dismutases (SOD for short)in human tissues. These include the cytoplasmic Cu/Zn superoxidedismutases and the mitochondrial manganese superoxide dismutase (MNSODfor short). These catalyze the decomposition of superoxide free radicals(O₂ ⁻), producing hydrogen peroxide (H₂O₂) which can in turn bedecomposed by catalases and/or glutathione peroxidases to H₂O and O₂.

It has been possible to show that the development of colorectal tumorsand hepatic metastases thereof is associated with a significant increasein MNSOD expression (Janssen et al. J. Cancer Res Clin. Oncol. 125(6),327-35, 1999). It was also possible to show this for lung tumors(Chung-man H J, et al. Cancer Research 1; 61(23), 8578-85, 2001) and forbreast cancer cells (Zhongkui Li et al., Free Radical & Medicine 30;260-267, 2001). It was observed in clinical studies that an increasedMNSOD antigen level in colorectal carcinomas, in stomach tumors and inglioblastomas is an independent prognostic factor for the reducedsurvival rate of the patients investigated (Janssen A M L et al. Br. J.Cancer, 78(8) 1051-1057, 1998; Janssen A M L et al. Clinical CancerResearch vol. 6., 3183-3192, 2000; Ria F. et al. British Journal ofCancer 84(4) 529-534, 2001). On the other hand, epithelial cells fromcarcinomas in situ of the breast and benign hyperplasias were more oftenfound to be strongly positive for MNSOD expression than neoplasticepithelial cells from invasive carcinomas of the breast (Soini Y. et al.J Pathol Sep.195(2),156-62, 2001).

Thioredoxin reductase (TXNRD for short) is a key enzyme for regulatingthe intracellular redox state. This enzyme catalyzes the NADPH-dependentreduction of thioredoxin disulfide and a large number of other oxidizedcellular constituents (Becker K, et al. Eur. J. Biochem. 267, 6118-6125,2000). Constitutive expression of TXNRD has been detected in varioushuman cell types, e.g. leukocytes. According to recent studies, TXNRDexpression is thought to be involved in the development of tumors(Söderberg A. et al. Cancer Research 60, 2281-2289, 2000). Glutathioneperoxidase (GPX for short) plays an important part in protection fromoxidative stress. This enzyme catalyzes the decomposition of H₂O₂ to H₂Oand O₂. Overexpression of GPX1 is therefore able to protect cells fromoxidative destruction and appears to be important especially when MNSODis also overexpressed, because accumulation of H₂O₂ is otherwisepossible (Li S., et al. Cancer Research 60, 3927-3939, 2000). A reducedGPX1 expression was observed in imexonresistant RPM/8226/I myeloma cells(Dvorakova K. et al. Molecular Cancer Therapeutics 1, 185-195, 2002).

It has been possible in recent years, through the identification andcharacterization of disseminated cancer cells, to achieve astonishingadvances in the diagnosis, prognosis and therapy of cancers. Thisapproach is based on the realization that the disseminated cancer cellsare a tumor entity independent of the primary tumor and therefore arefundamentally different from cells of the primary tumor on the basis ofa different genotype and phenotype. Thus, for example, it is possiblewith the aid of multiparameter analyses to answer, irrespective of thestatus of the primary tumor, questions with prognostic and therapeuticrelevance in a number of patients with breast cancer (Giesing M. et al.,The International Journal of Biological Markers vol. 15 (1), 94-99,1999).

One object of the present invention is to indicate a further practicablemethod permitting reliable cancer diagnosis. The method oughtadvantageously also to answer prognostic questions about the furthercourse of a cancer. A further object of the present invention is toindicate targets for medical treatment of cancer.

The present invention relates to a method for investigating biologicalsamples for cancer cells, where the expression of at least 2 genes whichare selected from

-   -   i) manganese superoxide dismutase genes;    -   ii) thioredoxin reductase genes; and    -   iii) glutathione peroxidase genes        is determined on at least one cell-containing fraction of the        biological sample.

The term cancer cell represents according to the invention a cell whichexhibits one or more modification associated with cancer, that isdysplasia in the general sense. The basis for this definition is theidea that a continuous alteration process is involved in the developmentof cancer. For example, a plurality of alterations, especially in thegenetic material or in the expression of the genetic material by cellsis usually required-to progress from a normal cell to a cancer cell andin particular to a tumor cell. The term cancer cell therefore alsoincludes precursors of cancer and in particular tumor cells withcancerous or tumorous modifications.

The gene expression analysis of the invention comprises determination ofthe expression of at least two genes (parameters). Analysis of a singleparameter essentially involves three method steps:

a) expedient provision of the gene expression product to be determined;

b) quantification of the gene expression product;

c) evaluation.

Method steps a), b) and c) are advantageously carried out in the statedsequence. Investigation of a plurality of parameters can take place inseparate methods or, in a preferred embodiment of the present invention,at least partly in parallel in an appropriately designed method, inwhich case at least method steps a) and b) are carried out in parallelfor at least 2 of the parameters i), ii) and iii) of the invention.

In a particular embodiment of the method of the invention, theexpression of at least one MNSOD gene is determined in combination withthe expression of at least one further gene selected from thioredoxinreductase genes and glutathione peroxidase genes. Of these, thecombination of MNSOD genes with TXNRD genes is preferred.

In a further particular embodiment of the method of the invention, theexpression of at least one MNSOD gene, of at least one TXNRD gene and ofat least one GPX gene is determined.

a) Provision of the gene expression product to be determined

The method of the invention is suitable for investigating samples of anybiological origin. One embodiment relates to body samples of human andanimal origin. Samples such as tissues, native, frozen, fixed, with andwithout dissection, blood and blood constituents or isolates thereof,further body fluids, e.g. bone marrow, lymph, sputum, lavages, puncturefluids, ascites, mucosal smears, exudates and urine, or stool, andespecially cell-containing fractions thereof, can advantageously beinvestigated by the method of the invention. It is accordingly an invitro method.

In a preferred embodiment of the present invention, body fluids,especially blood and blood constituents or isolates thereof, and alsobone marrow, in which cancer cells are present where appropriate, areinvestigated. Body fluids are investigated in particular fordisseminated cancer cells.

The term “disseminated cancer cell” is defined in particular in relationto solid tumors, that is to say in particular primary tumors, metastasesand recurrences. In contrast to solid tumors, disseminated cancer cellsare able to circulate in the body of an individual. This usually takesplace via endogenous transport organs, especially body fluids, inparticular blood. Disseminated cancer cells are usually derived from asolid tumor by initially being part of a solid tumor, that is inparticular of the tumor tissue, from which they subsequently becomedetached. In this way, disseminated cancer cells leave the region of thebody defined by the solid tumor, especially the morphological structuralunits affected by the tumor, for example the organ, and reach inter aliasites with which there is no morphological connection starting from thesolid tumor.

According to a particular aspect, disseminated-cancer cells arecharacterized by their relatively small amount in a sample based on thenon-cancer cells which are likewise present, i.e. they usuallyconstitute a comparatively small proportion of the cellular constituentsof the sample. They are therefore also referred to as residual cancercells (minimal residual disease, MRD for short). Considering for examplecell-containing body fluids, the proportion of disseminated cancer cellsis usually below 1:1000, mostly below 1:10000 and in many case evenbelow 1:100000, based on the number of non-cancer cells in a randomlyobtained sample of the body fluid. In the case of blood, these ratiosapply in particular in relation to mononuclear cells (for short: MNC).

Disseminated cancer cells are usually investigated in cell-containingmixtures which optionally comprise disseminated cancer cells in additionto non-cancer cells. The mixtures may comprise various proportions ofdisseminated cancer cells for the gene expression determination to becarried out according to the invention. However, proportions of at least50% cancer cells are expedient, proportions of at least 70% arepreferred and proportions of at least 80% are advantageous.

With a view to the gene expression analysis to be carried out accordingto the invention, if necessary a preparatory processing of the cellularconstituents present in the sample, and in particular of the geneexpression products to be determined, takes place, by which means thelatter are provided in an expedient form in relation to the method ofthe invention. Such processing usually corresponds to customary practiceand is based in particular on the requirements for expressiondetermination by protein or nucleic acid analysis.

An enrichment of cancer cells, and in particular of disseminated cancercells, going beyond this can likewise take place in a manner known perse, for example by known methods for isolating cancer cells, such asimmunospecific adsorption methods, microdissection methods, densitygradient methods or filtration methods.

Isolation means for the purposes of the present invention any enrichmentof a constituent to be isolated from a mixture which comprises thisconstituent in addition to at least another one. The result of theisolation may therefore perfectly well be a further mixture which,however, comprises the constituent to be isolated in a higherconcentration in relation to at least one other constituent comparedwith the original mixture.

According to a particular aspect, the processing of the invention takesplace with enrichment of cancer cells. This aspect relates in particularto the investigation of body fluids with relatively small proportions ofdisseminated cancer cells, in particular those described above. The aimof this type of processing is to provide test cells or, usually, a testcell mixture which then have or has a higher proportion of cancer cellsthan the original cells or the original cell mixture if cancer cells arepresent in the original cells or in the original cell mixture. Theoriginal cells, the original cell mixture or parts thereof may serve ascomparison cells or comparison cell mixture which then have or has alower proportion of cancer cells than the test cells or the test cellmixture if cancer cells are present in the original cell mixture.

A particular method for enrichment of disseminated cancer cells isdescribed in WO 00/06702. This method is incorporated in the presentdisclosure by reference. The disseminated cancer cells which can beenriched by this method are distinguished by their dedifferentiated,premetastatic character. They are therefore also referred to asmicrometastases. In contrast to disseminated cancer cells which can beenriched in particular with immunospecific adsorption methods, i.e. inparticular epithelial, relatively small cancer cells (especially withdiameters of about 20 μm or less), the cancer cells which can beenriched by the method described in WO 00/06702 have undergone anepithelial-mesenchymal transition: they are usually larger (inparticular with diameters of more than about 20 μm) and usually nolonger exhibit the organotypical expression pattern and/or theepithelial expression characteristics of the disseminated cancer cellswhich can be enriched in particular by immunospecific adsorptionmethods. Thus, whereas with epithelial disseminated cancer cells thereis still a certain connection to the primary tumor via the organotypicalexpression pattern and/or the epithelial expression characteristics, themesenchymal disseminated cancer cells are independent of the primarytumor. This makes them into disseminated cancer cells which arepreferably investigated according to the invention.

In the method described in WO 00/06702, a cell-containing body fluid orparts thereof, for example a nonspecifically enriched fraction, arepassed through a screen with a mesh or pore width of about 10 to 200 μm,and the screen residue remaining on the screen, i.e. the cell fractionretained on the screen, is obtained. A proportion of cancer cells of atleast 50% is achieved in the screen residue, as long as thecell-containing body fluid or parts thereof comprise cancer cells,through use of screens of particular mesh or pore size which make asize- and shape-dependent separation process possible. Screens used inthe known methods are sheet-like or porous structures with orificeswhich have dimensions such that non-cancer cells present in thecell-containing body fluid are able to pass through, whereas cancercells or cancer cell aggregates are retained.

In an advantageous further development of the method, which makes simpleautomation and standardization of the method possible, and at the sametime further increases the purity of the filtered cancer cell fraction,it is possible to use a flat filter with a mesh or pore width of about10-200 μm which is disposed in the filter housing which makes uniformrinsing through of the filter surface possible owing to a suitablefluidic design. This is described in particular in DE 100 54 632 and isincorporated in the present disclosure by reference.

Thus, in a particular embodiment, the cell-containing body fluid orparts thereof are conveyed into an inlet port of a filter housing, thebody fluid is passed laterally out of the inlet port into a fluidchamber on the inlet side of the filter housing and is distributed overa flat filter disposed in the filter housing and having a mesh or porewidth of about 10-200 μm essentially parallel to the surface of the flatfilter, the body fluid or the parts thereof are transported over theflat filter and separated into a residue remaining on the flat filterand a filtrate, the filtrate is collected in a fluid chamber on theoutlet side and is discharged through an outlet port and subsequentlythe residue is obtained.

If cancer cells are to be isolated from blood, it is preferred accordingto the invention initially to separate white blood cells by densitygradient centrifugation. Cancer cells are found in particular in thefraction which also comprises mononuclear cells, so that this fraction(referred to hereinafter as MNC fraction) is preferably passed on to thesubsequent filtration or alternatively to another method for isolatingcancer cells.

The filtration of the cell-containing body fluid or the fraction iscomplete when the total cell-containing fluid has passed through thescreen or the flat filter. A washing step may follow, in which furtherliquid, preferably buffer or culture medium, is passed through thescreen or the flat filter. The washing liquid can be added to thepreviously obtained filtrate or else be collected separately therefromand discarded where appropriate.

The cell fraction retained on the screen or flat filter can be passed ondirectly to the subsequent expression analysis or initially to storage.The residue comprising the cancer cells is advantageously initiallydetached from the screen or flat filter and collected. Variousprocedures can be chosen for this purpose depending on the nature of thesubsequent use.

For example, the residue can be incubated in a solution which leads tolysis of the cells and permits cellular constituents such as nucleicacids, proteins or lipids to be obtained. It is advantageous in thiscase for the solution to be agitated during the incubation. If themethod of the invention is carried out manually, it is possible forexample to connect a syringe piston in each case to the inlet port andto the outlet port of the filtering apparatus and to pump the solutionbackwards and forwards between the two syringes. If the method proceedsautomatically, a corresponding agitation of the solution can be achievedby conveying means such as, for example, pumps.

Vital cancer cells are obtained by dissolving the residue adhering tothe flat filter, advantageously by back-flushing the filter with aliquid which is conveyed from the fluid chamber on the outlet side ofthe filter housing into the fluid chamber on the inlet side. Theback-flushing liquid is advantageously a buffer solution or a culturemedium. The cancer or tumor cells obtained in this way can for examplebe cultivated to obtain cellular constituents or vaccines.Alternatively, cancer cells can be detached from the filter surfaceusing centrifugal force or by means of so-called optical tweezers.

Suitable screens or flat filters usually have a mesh or pore width ofabout 10-200 μm, preferably of 15 to 30 μm, particularly preferably of17-27 μm and very particularly preferably of about 20 μm. The flatfilter is advantageously designed as membrane filter, in which casetypical filter materials such as plastics networks or fabrics,microporous membrane filters, filter tile or combinations thereof can beemployed. Suitable filter materials and suitable methods for producingsuch filters are described in particular in WO 00/06702. It isparticularly preferred to use filters which are made ofsolvent-resistant material and which may consist for example of plasticssuch as polyethylene, polypropylene, polytetrafluoroethylene, highlyfluorinated polymers, vinylidene fluoride, aminoplastics and, inparticular, polyester.

To select the screen or filter suitable in each case for isolatingparticular cancer cells, the skilled worker can obtain, in preliminaryexperiments with screens or filters of increasingly narrow mesh (forexample in the sequence 200 μm, 115 μm, 74 μm, 51 μm, 38 μm, 30 μm, 27μm, 20 μm, 17 μm, 15 μm and 10 μm), individual cell fractions andinvestigate them for their therapeutic and diagnostic relevance. It mayalso prove advantageous in this connection to employ filtercombinations, i.e. in the above example for instance to filter off,using a 115 μm filter, less relevant, larger aggregates and to analyzeonly the cancer cell fraction collected on a downstream 20 μm filter.

The expression analysis of the invention relates to the determination ofany gene expression products such as proteins or nucleic acids and, inthis connection, especially mRNA and the nucleic acids which can bederived therefrom, such as cDNA. Generally known methods can be appliedto obtain these gene expression products, where appropriate mixed withfurther cellular constituents. For nucleic acids in particular themethods and reagents known to be suitable for the area of isolating andpurifying nucleic acids will be used, for example a solution comprisingguanidine isothiocyanate and phenol (cf. Lottspeich F. and Zorbas H.(editors) Bioanalytik, Heidelberg; Berlin: Spektrum, Akad. Verl., 1998,in particular chapter 21). It is possible in particular for mRNA to beisolated in the form of poly A⁺ mRNA by means of oligo-dT columnchromatography or correspondingly equipped magnetic beads.

b) Quantification of the gene expression product

The methods which can be used to quantify the respective gene expressionproduct are primarily governed by the nature of the gene expressionproduct. Thus, it is possible in principle to employ all the methodsknown to be suitable for quantifying proteins and nucleic acids from theareas of protein analysis and nucleic acid analysis. From the area ofprotein analysis, mention may be made for example of enzymatic activityassays, immunological techniques, certain spectroscopic methods and massspectrometry, if necessary in combination with chromatographic orelectrophoretic separation methods. In order to ensure specificdetection of the expressed proteins, immunological methods willadvantageously be used, as described for example in the studiesdescribed at the outset on the expression of the MNSOD, TXNRD1 and GPX1genes.

The skilled worker is able in particular starting from the respectiveamino acid sequence to produce anti-bodies which are directed againstthe protein. It is possible for this purpose to use the entire proteinor fragments thereof (polypeptides) as immunogen, and to produce, in amanner known per se, polyclonal and monoclonal antibodies and, basedthereon by means of recombinant techniques, also humanized antibodies,and fragments thereof.

These antibodies can then be used in particular in quantitativeimmunoassays and immunoblot techniques, e.g. Western blotting. Bothdirect and indirect assays are suitable. Competitive immunoassays, i.e.the protein or polypeptide to be detected competes as antigen withlabeled antigen for antibody binding, are in particular. Sandwichimmunoassays are preferred, i.e. the binding of specific antibodies tothe antigen is detected using a second, usually labeled antibody. Theseassays may be designed to be both homogeneous, i.e. without separationinto solid and liquid phase, and heterogeneous, i.e. bound labels areseparated from unbound ones, for example by solid phase-boundantibodies. The various heterogeneous and homogeneous immunoassayformats can be assigned, depending on the labeling and method ofmeasurement, to particular classes, for example RIAs(radioimmunoassays), ELISA (Enzyme Linked ImmunoSorbent Assay), FIA(fluorescence immunoassay), LIA (luminescence immunoassay), TRFIA(time-resolved FIA), IMAC (immunoactivation), EMIT (Enzyme MultipliedImmune Test), TIA (turbidimetric immunoassay).

Of the mass spectrometric methods, particular mention should be made ofthe so-called SELDI method. This comprises the protein mixtures to beinvestigated initially being trapped on suitable surfaces, e.g. solidsupport surfaces with affinity for proteins, unwanted substances beingremoved if necessary from the surfaces, for example by washing withsuitable liquids, and subsequently determination being carried out byMALDI-TOF Laser Desorption/Ionization Time-Of Flight Mass Analysis).

The skilled worker is equally able to find nucleic acids which encodethis protein or parts thereof and provide suitable means for specificdetection thereof.

Thus, methods from the area of nucleic acid analysis which should beparticularly mentioned are those based on the specific binding with thenucleic acid to be determined. Included herein is in particular thespecific amplification of the nucleic acid to be determined or partswhich can be derived therefrom, e.g. determination of mRNA by means ofquantitative PCR, and/or specific hybridization thereof onto optionallyimmobilized probes (especially with the aid of nucleic acid arrays, alsocalled biochips), if necessary after previous specific or nonspecificamplification.

The term “manganese superoxide dismutase (MNSOD for short) according tothe invention refers to enzymes which catalyze the decomposition ofsuperoxide free radicals (O₂ ⁻) to form hydrogen peroxide (H₂O₂). Theseinclude in particular the enzymes which constitute enzyme class1.15.1.1.

Owing to differences in phylogenetic development, there is a certainspecies-dependent heterogeneity within this group of enzymes. Thedetermination will be directed at the particular MNSOD to be expected inthe relevant organism, depending on the individual to be investigated.In a particular embodiment of the present invention, the determinationis directed at MNSODs of human origin.

In addition to species-dependent variations, there are also usually foreach species polymorphic variants which have different amino acidsequences owing to allelic variation. Variations of this type havealready been described (Barra et al. (1984) J. Biol. Chem. 259:12595-12601; U.S. Pat. No. 5,246,847 (FIG. 1A); U.S. Pat. No. 5,260,204(claim 1); U.S. Pat. No. 5,985,633 (SEQ ID NO: 1); the 9Ala-9Valpolymorphism described in Stoehlmacher J et al. (2002) Oncol. Rep. 9:235-238). Reference is made to the MNSODs described in thesepublications in their entirety.

In a particular embodiment of the present invention, the expressionanalysis is directed at an MNSOD having the amino acid sequence SEQ IDNO:13.

Further useful directions for the MNSOD determination of the inventioncan also be found by the skilled worker from the nucleic acid sequencesindicated in the aforementioned publications. In addition, there arenumerous entries in relevant gene databases for MNSOD-encoding nucleicacid sequences, on the basis of which the skilled worker is able toprovide suitable means for the sequence-specific detection of thesesequences and of expression products which can be derived therefrom.Mention should be made in particular in this connection of MNSOD mRNAwhich can be isolated from human liver tissue (Accession No. X14322),MNSOD mRNA which can be isolated from human colonic carcinoma (AccessionNos. X59445, X15132, Y00985 and M36693), MNSOD mRNA which can beisolated from human placental tissue (Accession No. X07834), cDNA whichcan be derived from a human T-cell DNA gene library (Accession No.E01408, cf. also JP 1987289187-A1), and DNAs and RNAs encoding variousMNSOD variants (Accession Nos. E03557, E08013 and E08014; cf. also JP1992117288-A1 and JP 1994245763-A1).

In a further particular embodiment, the sequence-specific detection ofMNSOD expression is directed at determination of an mRNA orcorresponding cDNA having the sequence SEQ ID NO:14 or a partialsequence thereof.

Specific amplification of this sequence is possible for example usingthe primer sequences of SEQ ID NO:1 and SEQ ID NO:2. A suitable probe isindicated for example by SEQ ID NO:3. This probe is particularlysuitable for the 5′-exonuclease detection using the two afore-mentionedprimer sequences.

The term “thioredoxin reductase” (TXNRD for short) according to theinvention refers to enzymes which catalyze the NADPH-dependent reductionof thioredoxin-S₂ to thioredoxin-(SH)₂. These include in particular theenzymes which constitute enzyme class 1.6.4.5.

An additional point is that the thioredoxin reductase family includes aplurality of thioredoxin reductase isoforms of which, besidesthioredoxin reductase 1, mention should be made in particular ofthioredoxin reductases of type 2 (e.g. α or β), or 3.

Owing to differences in phylogenetic development, there is a certainspecies-dependent heterogeneity within this group of enzymes. Thedetermination will be directed at the particular TXNRD to be expected inthe relevant organism, depending on the individual to be investigated.In a particular embodiment of the present invention, the determinationis directed at TXNRDs of human origin.

In addition to species-dependent variations, there are also usually foreach species polymorphic variants which have different amino acidsequences owing to allelic variation. Reference is made to the TXNRDsdescribed in these publications in their entirety.

In a particular embodiment of the present invention, the expressionanalysis is directed at a TXNRD1 having the amino acid sequence SEQ IDNO:15.

Further useful directions for the TXNRD determination according to theinvention can be found by the skilled worker from the nucleic acidsequences indicated in the aforementioned publications. In addition,there are numerous entries in relevant gene databases for TXNRD-encodingnucleic acid sequences, on the basis of which the skilled worker is ableto provide suitable means for the sequence-specific detection of thesequences and of expression products which can be derived therefrom.Mention should be made in particular in this connection of TXNRD mRNA(Accession Nos. AF106697, S79851, and AF201385), TXNRD mRNA which can beisolated from human placental tissue (Accession No. X9124), TXNRD mRNAwhich can be isolated from human brain tissue (Accession No. AF208018),TXNRD mRNA which can be isolated from human osteoblasts (Accession No.AJ001050), TXNRD1 mRNA which can be isolated from human large cell lungcarcinoma (Accession No. BC018122), TXNRD2α mRNA (Accession No.AB019694) and TXNRD2β mRNA (Accession No. AB019695) which can beisolated from human placental tissue, TXNRDβ mRNA which can be isolatedfrom human melanoma (Accession No. BC007489), TXNRD GRIM-12 mRNA whichcan be isolated from human breast carcinoma (Accession No. AF077367),TXNRD2 mRNA (Accession No. AF171055) and TXNRD3 mRNA (Accession No.AF133519 and AF171054).

In a further particular embodiment, the sequence-specific detection ofTXNRD expression is directed at determination of TXNRD1 expression andin particular of an mRNA or corresponding cDNA having the sequence SEQID NO:16 or a partial sequence thereof.

Specific amplification of this sequence is possible for example usingthe primer sequences SEQ ID NO:4 and SEQ ID NO:5. A suitable probe isindicated for example by SEQ ID NO:6. This probe is particularlysuitable for 5′-exonuclease detection using the two primer sequencesmentioned above.

The term “glutathione peroxidase” (GPX for short) according to theinvention refers to enzymes which—similar to catalases—catalyze thedecomposition of hydrogen peroxide (H₂O₂) to form water and oxygen.These include in particular the enzymes which constitute enzyme class1.11.1.9.

An additional point is that the glutathione peroxidase family includes aplurality of glutathione peroxidase isoforms of which, besidesglutathione peroxidase 1, mention should particularly be made of theglutathione peroxidases of type 2, 3, 4, 5 or 6.

Owing to differences in phylogenetic development, there is a certainspecies-dependent heterogeneity within this group of enzymes. Thedetermination will be directed at the particular GPX to be expected inthe relevant organism, depending on the individual to be investigated.In a particular embodiment of the present invention, the determinationis directed at GPXs of human origin.

In addition to species-dependent variations, there are also usually foreach species polymorphic variants which have different amino acidsequences owing to allelic variation. Variations of this type havealready been described; e.g. a Pro-Leu amino acid substitution atposition 197 (Forsberg L et al. (1999) Hum. Mutat. 14(4):294-300).Reference is made to the GPXs described in these publications in theirentirety.

In a particular embodiment of the present invention, the expressionanalysis is directed at a GPX1 having the amino acid sequence SEQ IDNO:17.

Further useful directions for the GPX determination according to theinvention can also be found by the skilled worker from the nucleic acidsequences indicated in the aforementioned publications. In addition,there are numerous entries in relevant gene databases for GPX-encodingnucleic acid sequences, on the basis of which the skilled worker is ableto provide suitable means for the sequence-specific detection of thesesequences and of expression products which can be derived therefrom.Mention should be made in particular in this connection of human GPXmRNA (Accession No. AF217787), exon 1, 2, and 3 to 5 human GPX DNA(Accession Nos. D16360, D16361 and D16362), GPX mRNA which can beisolated from human placental and fetal liver tissue (Accession No.D00632), GPX mRNA which can be isolated from human liver tissue(Accession Nos. Y00433 and E02175, cf. also JP 1990002362 A1), GPX mRNAwhich can be isolated from human renal tissue (Accession No. Y13710,Y00369, X13709 and X13430), GPX DNA which can be isolated from humanleukocytes (Accession No. Y00483), GPX1 mRNA which can be isolated fromhuman myelocyte leukemia cells (Accession No. M21304), GPX2 DNA which beisolated from human colonic carcinoma (Accession No. X91863), GPX2 mRNAwhich be isolated from human bladder carcinoma (Accession No. BC005277and BC016756), GPX2 DNA which be isolated from human fibroblasts(Accession No. AF199441), GPX3 mRNA which be isolated from humanplacental tissue (Accession No. X58295), GPX3 mRNA which be isolatedfrom human large cell lung carcinoma (Accession No. BC013601), GPX3 mRNAwhich be isolated from human spleen tissue (Accession No. BC025956),GPX4 mRNA which be isolated from human testicular tissue (Accession No.X71973), GPX4 mRNA which be isolated from human melanoma (Accession No.BC010157) and GPX5 mRNA which be isolated from human epididymis tissue(Accession No. AJ005277).

In a further particular embodiment, the sequence-specific detection ofGPX expression is directed at determination of GPX1 expression and inparticular of an mRNA or corresponding cDNA having the sequence SEQ IDNO:18 or a partial sequence thereof.

Specific amplification of this sequence is possible for example usingthe primer sequences SEQ ID NO:7 and SEQ ID NO:8. A suitable probe isindicated for example by SEQ ID NO:9. This probe is particularlysuitable for 5′-exonuclease detection using the two primer sequencesmentioned above.

The term amplification refers to the multiplication of nucleic acids,i.e. the generation of many copies of particular nucleic acids. Theamplification usually proceeds at least linearly and preferablyexponentially.

Known amplification methods can be used, which include the polymerasechain reaction (PCR), also carried out in principle as nested PCR,asymmetrical PCR or multiplex PCR, or alternative methods such as theligase chain reaction (LCR), nucleic acid sequence-based amplification(NASBA), transcription-mediated amplification (TMA) and the like.Certain versions of these techniques and/or combinations with othermolecular biology methods may be expedient.

The amplification procedure is preferably based on PCR techniques. Forthis purpose, usually at least two primers differing in polarity (i.e.at least one pair of primers composed of a forward primer and a reverseprimer) are used per template.

In a particular embodiment of the present invention, a pair of specificprimers is used per nucleic acid sequence to be determined. Anadditional possibility is to amplify the total RNA of a sample (cf., forexample, Zohlnhöfer D. et al. Circulation 103, 1396-1402, 2001) andsubsequently to determine particular RNAs as corresponding cDNAs byspecific hybridization.

In a particular embodiment of the present invention, at least one primerwhich is labeled is used for the amplification. The labeling is used todetect an amplicon into which the labeled primer has been incorporatedduring the amplification.

The skilled worker is aware of a large number of suitable labelstogether with relevant detection systems. Fluorescent and chemi- orbioluminescent labels are preferred for reasons of sensitivity andpractical handling.

Labeling systems which are suitable in principle are those which can bedetected for example spectroscopically, photochemically, biochemically,immunochemically, electrically, optically or chemically. These includeboth direct labeling systems such as radioactive markers (e.g. ³²P, ³H,125I, ³⁵S, ¹⁴C), magnetic markers, chromophores, for example UV-, VIS-,or IR-absorbing compounds, fluorophores, chemi- or bioluminescentmarkers, transition metals, which are usually chelate-bound, or enzymes,e.g. horseradish peroxidase or alkaline phosphatase and the detectionreactions coupled thereto, and indirect labeling systems, for examplehaptens such as biotin or digoxigenin, which can be detected byappropriate detection systems.

Advantageous chromophores have an intense color which is only slightlyabsorbed by surrounding molecules. Classes of dyes such as quinolines,triarylmethanes, acridines, alizarins, phthaleins, azo compounds,anthraquinones, cyanines, phenazathionium compounds or phenazoxoniumcompounds may be mentioned here as representative of the wide range ofchromophores suitable according to the invention.

Fluorescent labels are advantageous. Strong signals are obtained withlittle background, high resolution and high sensitivity. It is importantaccording to the invention that one and the same fluorophore may emit aplurality of different radiations depending on the excitation andprinciple of detection.

Fluorophores can be used alone or in combination with a quencher (e.g.molecular beacons).

Examples of preferred fluorophores are aminomethylcoumarin acetic acid(AMCA, blue) EDANS, BODIPY 493/503; FL; FL Br2; R6G; 530/550; 558/568;TMR 542/574; TR 589/617; 630/650; 650/665, 6-FAM fluorescein (green),6-OREGON green 488, TET, Cy3 (red), rhodamines (red), 6-JOE, VIC, HEX,5-TAMRA, NED, 6-ROX, TEXAS Red7 (red), Cy5, Cy5.5, LaJolla Blue, Cy7,Alexa Fluor carboxylic acids, especially of the 647 and 532 type, e.g.as succinimidyl ester, and IRD41.

Particularly preferred fluorophores are Cy5, 5-Tamra and Cy3, and AlexaFluor carboxylic acids.

Chemiluminescent or bioluminescent labels are likewise advantageous.Preferred labels of this type are based for example on reactions ofalkaline phosphatase with dioxetane phosphate (AMPPD) or acridiniumphosphate substrates; of horseradish peroxidase with luminol oracridinium ester substrates; of microperoxidases or metal porphyrinsystems with luminol; of glucose oxidase, of glucose-6-phosphatedehydrogenase; or of luciferin/luciferase systems.

A label can in principle be introduced in any manner into an amplicon tobe detected as long as it permits detection thereof. A distinction canbe made in principle between direct and indirect labeling. With directlabeling, the detectable label is incorporated during the amplification.With indirect labeling there is initial incorporation of a primary labelwhich has a certain affinity for the detectable label which is to beadded subsequently. The latter procedure is always advantageous when thelabel to be used might influence the course of the amplification. Theindirect procedure is preferred especially in the case of chemi- orbioluminescent labels. Biotin/streptavidin systems in particular haveproved to be expedient according to the invention. Accordingly, thelabeled primers to be used according to the invention in a particularembodiment of the present invention are labeled with biotin ordigoxigenin, preferably at the 5′ end.

If at least two different nucleic acids are determined according to theinvention, it is usually advantageous to carry out a so-called multiplexamplification, i.e. multiplex PCR in particular. The intention in thiscase is to subject at least two different nucleic acids to the specificamplification, this taking place in a joint approach. In particular, themultiplex PCR with the aid of at least two, in each case specific, pairsof primers generates just as many different amplicons as long as theappropriate templates are present. It is possible in this case for theprimers or pairs of primers to be configured according to the abovestatements. It is particularly preferred according to the invention forall the primers or pairs of primers to be configured in the same way, sothat the generated amplicons can all be treated in an analogous mannerin the subsequent method steps. In particular, it is advantageous to beable to detect the amplicons in a joint method step.

In addition, such a multiplex approach may also include furtheramplification systems which may serve in particular as a control. Thus,it may in particular be desired also to include standardization,mismatch, housekeeping, sample preparation, hybridization oramplification controls.

It is particularly preferred according to the invention to determine themRNA transcribed by the relevant genes. This may involve mRNA which isalready spliced or not yet spliced. The determination is advantageouslydirected at spliced mRNA.

Ordinarily it is not the mRNA which is directly detected but nucleicacids which be derived therefrom. This applies especially in the casewhere the detection includes an amplification by PCR. For this purpose,the mRNA is normally initially transcribed into DNA. This is possiblefor example by reverse transcription, in which case the DNA (cDNA)complementary to the mRNA is generated. Suitable procedures forgenerating cDNA from mRNA are familiar knowledge in the art.

The resulting cDNA can subsequently be quantified by PCR as a measure ofthe amount of corresponding mRNA present. Suitable methods for this arealso known to the skilled worker (see, for example, Chapter 24.2.5 inLottspeich F. and Zorbas H. (editors) Bioanalytik, Heidelberg; Berlin:Spektrum, Akad. Verl., 1998, in particular Chapter 21). The techniqueknown by the specialist term of “5′-exonuclease assay” has proved to beparticularly effective in this connection. In this case, a labeled probewhich is annealed between the two primers onto the nucleic acid sequenceto be detected (template), and is degraded during the primer extensionthrough the 5′-3′-exonuclease activity of the polymerase used, is used.The degradation generates a signal. Detection of this signal can beregarded as a measure of the progress of amplification, because thestrength of the signal is proportional to the number of ampliconsgenerated, and the time-dependent change in the signal additionallypermits conclusions to be drawn about the amount of template. This typeof assay is commercially available under the name “TaqMan®”.

A further possibility for detecting nucleic acids is provided bytechniques based on specific hybridization. These techniques can inprinciple be applied to the nucleic acids present in the sample, andnucleic acids which can be derived therefrom, it being possible for thenucleic acids, or the nucleic acids which can be derived therefrom, tobe subjected to an amplification beforehand, or not.

A large number of different hybridization formats are known in principleto the skilled worker. It is preferred according to the invention to useprobes for sequence-specific nucleic acid detection. The procedure forthis is usually as follows.

If the nucleic acid to be detected results in double-stranded form, itshould be converted into a single-stranded form beforehand. Suitablemeasures are sufficiently well known to the skilled worker. Thus,double-stranded nucleic acid as generated for example by a PCR can besubjected to denaturing conditions, such as elevated temperature, highionic strength and/or alkaline pH.

For the hybridization, the hybridization components are allowed to acton one another under conditions which allow duplex formation betweennucleic acid sequence to be detected and probe complementary thereto.For example, the immobilized probe is brought into contact with ahybridization mixture which includes the nucleic acid or a nucleic acidwhich can be derived therefrom and, where appropriate, further customaryadditions. It is self-evident that parts of the mixture can initially bebrought into contact separately from one another with the probe. Thehybridization conditions are expediently chosen so that probe and targetcomplementary thereto are able to form stable hybrids. Conditions ofrelatively low stringency are usually chosen initially, e.g.temperatures of about 20-50° C. and ionic strengths of about 6×SSPE orlower. Subsequent washing is then possible at similar or higherstringency e.g. about 2×SSPE to about 0.1×SSPE at about 30-50° C. It isalso possible to have recourse to known agents, e.g. detergents,blocking reagents, denaturing agents, agents which acceleraterenaturation and Tm-equalizing agents. Optimization of the hybridizationprotocol is a matter for the skilled worker.

Immobilized probes are preferably used for the hybridization. For thispurpose, the probes are coupled to a support, for example by covalent,adsorptive or by physical/chemical interactions of probe and surface.Suitable methods for achieving an expedient coupling are known to theskilled worker. It is moreover possible for the previously preparedprobe to be coupled to the surface, or for the probe to be synthesizedin situ on the surface, e.g. by means of photolithographic methods.Coupling via photoactive groups, for example certain anthraquinones, andif necessary a spacer of suitable length and constitution, for examplepolyethylene glycol with n=2-10 and preferably of about 6 for 8-60 mers,to a reactive surface represents a preferred embodiment. Arrays ofimmobilized probes are particularly preferred as convenient andefficient format. Reference is made to the statements concerning this inWO 02/18634 in their entirety.

The sequence-specific detection is usually based on determining whetherthe probe and nucleic acid form a duplex, i.e. hybridize together. Thedetection according to the invention thus comprises whether a particulartarget sequence can be found or not (presence or absence). Thedetermination is to take place quantitatively in relation to theexpression products of the MNSOD, TXNRD and GPX genes.

The detection usually requires quantification of the nucleic acids whichhybridize onto an immobilized probe. The quantification can take placeabsolutely or relatively. Suitable detection systems are sufficientlywell known to the skilled worker. A frequently used possibility consistsof introducing labels, e.g. of a radioactive, calorimetric, fluorescentor luminescent nature. These are introduced according to the inventionpreferably during an amplification which precedes the hybridization—asalready explained above—or detected, in the case of indirect labelingafter introduction of a primary label such as biotin or digoxigenin(DIG), by adding appropriate markers such as fluorescence-labeled orPOD-labeled streptavidin or anti-DIG and, in the case of chemi- orbioluminescence, normally by addition of enzyme substrate solutions or,if substrate molecules such as luminol are used as markers, of enzymesolutions.

The skilled worker is able to choose from the large number of suitabledetection systems in particular the photoelectric area sensors describedin WO 02/08458, in particular CCD cameras, or the fluorescence scannersdescribed in DE 100 38 080.

c) Evaluation

It is possible with the measurement methods described above to assign toeach investigated sample a particular value which characterizes theexpression of the investigated gene. It is particularly importantaccording to the invention to determine whether expression in cells ofthe investigated sample is comparatively elevated, because an elevatedMNSOD, TXNRD and GPX expression is relevant to cancer. The evaluationaccording to the invention therefore usually includes a comparison withcells in which no cancer-associated modification is to be expected(non-cancer cells, normal cells). If the investigation according to theinvention is directed for example at cancer cells in body fluids, thecells chosen for comparison will be those normally occurring in thisbody fluid. In the case of blood, these are in particular the whiteblood cells which can be obtained for example by density gradientcentrifugation (e.g. the buffy coat; the MNC fraction) or be separatedby more specific isolation methods (e.g. CD45-positive lymphocytes).These white blood cells can in principle also serve as comparison cellsor comparison cell mixtures when investigating body fluids other thanblood. A further possibility is for at least one cell-containingfraction of the sample, in particular of the body fluid, to befractionated by procedures for enriching cancer cells into at least twopartial fractions, of which one fraction, optionally enriched in cancercells, can be used as test cell mixture, and the other fraction,optionally depleted in cancer cells, can be used as comparison cellmixture.

If the investigation according to the invention is carried out on cellmixtures, it is not required for these mixtures to comprise exclusivelycancer cells or exclusively non-cancer cells. On the contrary, theimportant point is the ratio of the cell types to one another in themixtures. It is sufficient for the method of the invention if theproportion of cancer cells which is to be expected on the basis of theprocedures for obtaining the mixtures is significantly higher in thetest cell mixture than in the comparison cell mixture. Thus, proportionsof cancer cells are perfectly possible in the comparison cell mixture,as long as the proportion of cancer cells in the test cell mixture issufficiently higher. This can be ensured for example by obtaining thetest cell mixture from the comparison cell mixture by procedures forenriching cancer cells.

In a preferred embodiment of the present invention, therefore, a firstcell-containing fraction is obtained from the biological sample withenrichment of cancer cells, and the expression of the genes in thecell-containing fraction is determined, a further cell-containingfraction of the biological sample or of a comparable biological sampleis prepared, and the expression of the genes in the furthercell-containing fraction is determined, and the expression of each genein the cell-containing fraction is compared with its expression in thefurther cell-containing fraction. It is advantageous for the comparablebiological sample to be derived from the individual whose biologicalsample is investigated for cancer cells, i.e. a comparison is made withthe patient's own non-cancer cells. This is particularly important whenthe relevant patient has already received therapeutic procedures whichhave effects on the phenotype and genotype of his cells.

The test principle according to the invention is therefore based ondetermining whether enrichment of cancer cells is associated with ameasurable increase in MNSOD, TXNRD and GPX expression. The ratio of theexpression measured in the test cell mixture to the expression measuredin the comparison cell mixture is therefore decisive.

It will usually be expedient for validation of a particular test systemto fix a particular quotient (limit) above which overexpression ispresent by definition.

This limit may depend on the cell mixtures used and, in particular, onthe obtaining thereof. Thus, it is expedient to carry out a particularembodiment of the method of the invention initially on healthyindividuals, i.e. not suffering from cancer, and to fix, with the aid ofstatistical methods, a suitable limit for the method embodiment used.Thus, it is possible, taking account of statistical significance, tocarry out the method on a sufficiently large group of individuals, toform the average from the measured expression ratios, and to fix thelimit taking account of the average and of the relevant standarddeviation. A limit found in this manner also takes account for exampleof the cases in which the test cell mixture shows enhanced expression ofthe measured parameter by comparison with the comparison cell mixture,although the test cell mixture does not contain any cancer cells either.Such cases may occur in particular when the method chosen actually forenrichment of cancer cells leads to enrichment of non-cancer cells whichlikewise show enhanced expression of the measured parameter. This casewas observable for example in relation to GPX1 expression when the whiteblood cells were subjected to a size- and shape-dependent separationprocess.

It is moreover usually expedient to relate the values measured on thetest cell mixtures and comparison cell mixtures to a standard. Such astandard can be produced for example with the aid of cell lines whichshow a sufficiently strong expression of the gene expression product tobe determined (positive control). For example, the breast carcinoma cellline EFM 192 is suitable as positive control for determining MNSOD andGPX1 expression, and the breast carcinoma cell line MES-SA/Dx5 issuitable for example as positive control for TXNRD1 expression. Furthersuitable reference cell lines are either known or can be established bythe method of the invention, such as, for example, the breast carcinomacell line BT474.

The investigation according to the invention for cancer cells includesin particular identification thereof and/or characterization thereof.

A further aspect of the present invention is therefore the use of themethod of the invention for identifying cancer cells, in particular inearly diagnosis of tumors. This is connected in particular with theanalytical finding of whether the investigated sample has cancer cells,or the diagnostic finding of whether the individual whose sample hasbeen investigated is suffering from cancer. An elevated expression of atleast one MNSOD gene in combination with an elevated expression of atleast one TXNRD gene and/or at least one TGTPX gene, and in particularan elevated expression of at least one MNSOD gene in combination with anelevated expression of at least one TXNRD gene in combination with anelevated expression of at least one GTPX gene is to be regarded as anindication of the presence of cancer cells in the investigated samples.

This includes in particular detection of tumor cells from sputum/saliva,especially for early diagnosis of lung tumors; from urine, especiallyfor early diagnosis of prostate and bladder tumors; from stool,especially for early diagnosis of colonic and pancreatic tumors; fromblood/bone marrow/lymph, especially for early diagnosis of alldisseminating tumors.

One aspect of the present invention is also the use of the method of theinvention for characterizing cancer cells, e.g. for classifying tumorsand for estimating the risk for the patient. This is connected withprognostic findings about the future course of a cancer, such as theprobability (risk) of developing a metastasis or a recurrence, or ofsurviving a particular time, and therapeutic findings about the efficacyof an applied therapy (therapy monitoring) or findings for choice of thetherapy. An elevated expression of at least one MNSOD gene incombination with an elevated expression of at least one TXNRD geneand/or of at least one TGTPX gene, and in particular an elevatedexpression of at least one MNSOD gene in combination with an elevatedexpression of at least one TXNRD gene in combination with an elevatedexpression of at least one GTPX gene is associated with an increasedrisk of developing a metastasis or a recurrence, and with a reducedprobability of surviving a particular time. In order to assess theefficacy of an applied therapy (therapy monitoring), the method of theinvention is carried out on at least two different dates, i.e. beforeand after a particular therapeutic procedure. It is possible todetermine by comparing the expression determined before and after theprocedure whether the therapeutic procedure has led to a change in thenumber of cancer cells identifiable by the method of the invention inthe sample. A decrease is an indication of the efficacy of thetherapeutic procedure. It is possible in this way to assess inparticular those therapeutic procedures intended to reduce or eliminatedisseminated cancer cells.

In a particular embodiment, the method of the invention is part of amultiparameter investigation which, in addition to the three geneexpression analyses of the invention, also includes the investigation offurther parameters. In principle, as many parameters as possible shouldbe investigated, so that the only reasons for a restriction are usuallythose of expediency and practicability. Such multiparameterinvestigations usually involve investigation of up to 10000, inparticular up to 1000, preferably up to 100, particularly preferably upto 75, 50 or 25 and in particular up to 10, parameters.

The term “parameter” refers in this connection to any biochemical ormolecular biological peculiarity of cancer cells and in particular ofdisseminated cancer cells. Included herein are both therapeutic anddiagnostic, especially prognostic, parameters. Genomic parameters, e.g.expressed at the DNA level, are included therein just as much asparameters from the area of expression, e.g. those expressed at the RNA,in particular mRNA, or protein levels. Examples of possible parametersare mutations, insertions, deletions, LOHs, amplifications, aberrationsin the set of chromosomes and the like; the expression of splicevariants; and the over—and underexpression of certain mRNAs orproteins—and further unusual, in particular cancer-specific, alterationsof particular cellular constituents.

Preferred parameters are those relating to qualitative peculiarities ofthe DNA and/or RNA apparatus. Parameters which should be particularlymentioned in this connection are those relating to cellular propertiessuch as cell division, cell growth, cell-cell interactions, inhibitionof tumor suppression and therapy resistances, and especially havingoncogenic influences and thus also determining the clinical picture of acancer. Parameters from the area of DNA recombination, DNAamplification, DNA repair, cell cycling inducers and apoptosisinhibitors are particularly included therein.

Examples which may be mentioned are:

-   -   especially oncogenes and tumor suppressor genes, such as p53,        genes of the ras family, erb-B2, c-myc, mdm2, c-fos, DPC4, FAP,        nm23, RET, WT1 and the like, LOHS, for example in relation to        p53, DCC, APC, Rb and the like, and BRCA1 and BRCA2 for        hereditary tumors, microsatellite instability of MSH2, MLH1, WT1        and the like; also tumorous RNAs, such as CEA, cytokeratins,        e.g. CK20, BCL-2, MUC1, especially tumor-specific splice        variants thereof, MAGE3, Muc18, tyrosinase, PSA, PSM, BA46,        Mage-1 and the like, or else morphogenic RNAs, such as maspin,        HCG, GIP, motilin, hTG, SCCA-1, AR, OR, PR, various hormones and        the like;    -   in addition especially RNAs and proteins which relate to the        profile of metastasis, i.e. the expression of angiogenesis,        motility, adhesion and matrix degradation molecules such as        bFGF, bFGF-R, VEGF, VEGF-Rs such as VEGF-R1 or VEGF-R2,        E-cadherin, integrins, selectins, MMPs, TIMPs, SF, SF-R and the        like, to the cell cycle profile or proliferation profile, such        as cyclines (e.g. the ratio of cyclin D, E and B expressions),        Ki67, P120, p21, PCNA and the like, or the apoptosis profile        such as FAS (L+R), TNF (L+R), perforin, granzyme B, BAX, bcl-2,        caspase 3 and the like.

These and further parameters are described and explained in WO 99/10528,WO 00/06702 and in Giesing M. et al., The International Journal ofBiological Markers Vol. 15(1), 94-99, 1999. These statements areincorporated in this description in their entirety by reference.

The present invention also relates to analysis kits for carrying out themethod of the invention. These usually comprise

-   i) at least one means for determining MNSOD gene expression, in    particular specific antibodies or, preferably, sequence-specific    primers and/or probes like those described above, e.g. primers    having the sequences SEQ ID NO:1 and/or SEQ ID NO:2, or probes    having the sequence SEQ ID NO:3;-   ii) at least one means for determining TXNRD1 gene expression, in    particular specific antibodies or, preferably, sequence-specific    primers and/or probes like those described above, e.g. primers    having the sequences SEQ ID NO:4 and/or SEQ ID NO:5, or probes    having the sequence SEQ ID NO:6; and/or-   iii) at least one means for determining GPX1 gene expression, in    particular specific antibodies or, preferably, sequence-specific    primers and/or probes like those described above, e.g. primers    having the sequences SEQ ID NO:7 and/or SEQ ID NO:8, or probes    having the sequence SEQ ID NO:9; and, where appropriate,-   iv) further usual means for carrying out the method of the    invention.

Further particular embodiments of kits of the invention are evident fromthe statements about the method itself.

A further aspect of the present invention relates to a method for thetesting and/or functional validation of active substances. For thispurpose, the active substance is usually allowed to act ex vivo ondisseminated cancer cells which are characterized by elevated expressionof the MNSOD, TXNRD1 and/or GPX1 genes, and the response thereof isdetermined, in particular the expression of the MNSOD, TXNRD1 and/orGPX1 genes resulting after the action of the active substance. Ascontrol, the procedures of the method can be carried out in acorresponding manner on cells whose expression of the MNSOD, TXNRD1and/or GPX1 genes is not elevated. It is usually possible to haverecourse to cytobiological test systems known per se. If necessary,disseminated cancer cells can be maintained in culture and suitablebioassays can be carried out. For example, it is possible in this way totest known active substances with an antineoplastic effect and/or activesubstances employed for adjuvant therapy. It is possible in particularto test targeted active substances. These targets may be according tothe invention in particular the MNSOD, TXNRD1 and/or GPX1 genes or theexpression products thereof. However, other targets functionallyassociated with said genes and the expression products thereof are alsoconceivable and therefore can be validated in relation to MNSOD, TXNRD1and/or GPX1 gene expression. This is an important aspect of thedevelopment of active substances, according to which potential activesubstances can be selected in a targeted manner—for example by means ofscreening methods—and subsequently validated.

The purpose of this method for functional validation of activesubstances is to determine active substance-dependent, molecular and/ormorphological alterations in the disseminated cancer cells. Ifdetermination of one or more parameters on the disseminated cancer cellsreveals a state, after the action of the active substance, which differsfrom the state which existed before the action of the active substance,the target in relation to the active substance, or the active substancein relation to the target, is functionally validated according to theinvention. The functional validation detects in particular a functionalassociation between active substance and target in disseminated cancercells.

The functional validation of the invention on disseminated cancer cellsmay where appropriate be based on a functional prevalidation of thetarget on other cell systems. For example, targets can be cloned andexpressed in a manner known per se. Cell systems suitable for thispurpose, especially human cell lines, are available to the skilledworker and can be transfected appropriately. Such target-displaying cellsystems can be brought into contact in the manner already describedabove with one or more active substances. This method is also used toestablish a molecular and/or morphological action algorithm which can inturn be validated according to the invention on disseminated cancercells.

This method offers an advantageous basis for the development and testingof targeted active substances. The aiming at targets which have beenclinically and functionally validated uniformly on disseminated cancercells allows active substances to be developed with inclusion ofpharmaco- and toxicogenomic aspects to reduce unwanted side effects anda correct stratification of patients, i.e. an—if necessarytime-dependent—individualized use of active substances. Considerablesavings in costs and time result by comparison with conventional activesubstance developments.

A further aspect of the present invention relates to the treatment ofcancer by modulating the expression of at least two genes which areselected from manganese superoxide dismutase genes, thioredoxinreductase genes and glutathione peroxidase genes.

It is the particular purpose of the treatment according to the inventionto reduce the expression of these genes. In this case, the treatment isdirected mainly at the cellular constituents of body fluids in whichdisseminated cancer cells have previously been diagnosed. It is to beassumed that the phenotype of a majority of the disseminated cancercells present in an individual is influenced by the treatment accordingto the invention so that the ability of the disseminated cancer cells tosurvive is reduced.

Without being tied to a particular mechanism, such an effect oftreatment can be explained by the disseminated cancer cells losing aprotective mechanism which is mediated by overexpression of one or moreof the above genes, or being less adapted to the conditions prevailingin the particular body fluid, so that enhanced elimination of thedisseminated cancer cells occurs.

An advantageous treatment variant of the invention is directed atmodulation of MNSOD expression in combination with modulation of TXNRDand/or GPX expression. Modulation of MNSOD, TXNRD and GPX expression isparticularly advantageous.

Methods and means for modulating the expression of particular genes areknown in principle. In particular, gene expression can be reduced forexample at the RNA level with the aid of specific antisense molecules.At the protein level, expression is reduced with the aid of specificbinding partners which have a sufficient affinity for the expressedproteins and impair the function thereof. These include, for example,specific antibodies, but also low molecular weight compounds which, inthe present case, can be developed on the basis of the reactionscatalyzed by the enzymes and, in particular, the substrates converted.Accordingly, it is possible in particular to employ inhibitors of MNSOD,TXNRD and/or GPX activity.

An effective amount of a combination of active substances which is ableto reduce the expression of at least two genes selected from MNSOD,TXNRD and GPX genes, and to eliminate disseminated cancer cells in atreated individual is therefore administered according to the inventionto the individual to be treated.

The present invention therefore also relates to the use of a combinationof appropriate active substances for providing a pharmaceuticalcomposition for the treatment of cancer. In this connection, thiscombination of active substances can be administered in the form of anappropriate cocktail of active substances or in the form of individualactive substances at different times, for example alternately atdifferent times of day or sequentially, where the active substances areusually prepared as pharmaceutical composition in accordance with therules of pharmaceutical practice.

Exemplary Embodiments

DESCRIPTION OF THE FIGURE

FIG. 1 shows the evaluation, plotted as bar diagram, of a CCD contactexposure image of the fluorescence radiation emitted from an arraypopulated with the specified gene-specific probes after hybridizationwith cDNA single-stranded fragments obtained from cells in the blood ofa tumor patient.

SAMPLES

The following samples are used for the investigations described below:

Blood from 9 healthy donors and 47 tumor patients; breast carcinoma cellline BT474 (reference cell line for MNSOD, TXNRD1 and GPX1overexpression)

Tumor Cell Isolation (Cancer Cell Fraction C)

10 ml of heparinized blood are centrifuged (400 g; 10 min; RT). Thesupernatant plasma is removed. The pelleted cells are taken up in 12 mlof PBS. After density gradient centrifugation (Nycodenz 1.077; 800 g; 30min, RT) the interphase cells (essentially mononuclear cells, MNCfraction for short) are removed and washed 2× in 10 ml of PBS (1 mMEDTA) (400 g; 10 min; 4° C.). The MNC fraction is taken up in 10 ml ofPBS (1 mM EDTA, 0.5% BSA). 1 ml of this cell mixture is removed aspossible reference (comparative fraction A′). The remaining 9 ml of cellmixture are passed via a column through a screen woven from polyesterfilaments with a 20 μm mesh width (marketed by SEFAR AG, Rüschlikon,Switzerland), and the flow-through from the screen is collected aspossible reference (comparative fraction B′). The column is washed 5×with 10 ml of PBS (1 mM EDTA) each time. The screen is removed, invertedand incubated in a reaction vessel with 0.7 ml of Trizol® (5 min; RT).The screen is placed above the Trizol® solution in the reaction vesseland centrifuged (200 g; 30 s; RT). The dry screen is removed and theTrizol® solution (cancer cell fraction C) passed on for further RNSisolation.

An alternative possibility to incubation of the screen in Trizol® is forthe screen to be removed from the column, inverted and transferred intoPBS (1 mM EDTA, 0.5% BSA), the cells can be pelleted by centrifugation(400 g; 10 min, 4° C.) and passed on for further RNA isolation.

Normal Cell (Non-Cancer Cell) Isolation (Comparative Fractions A and B)

CD45-positiven lymphocytes are isolated as comparative fractions byremoving in each case 1/10 of the MNC fraction before (fraction A′) andafter (fraction B′) the screening process. They are transferred into areaction vessel containing 1 ml of PBS (0.5% BSA, 100 μg hu-IgG). 50 μlof washed anti-CD45 microbeads are added thereto. The mixture is rotatedat 4° C. for 20 min. The reaction vessel is then positioned on amagnetic strip in such a way that the microbeads (bound to CD45-positiveMNCs) are pelleted on the vessel wall. A pure population ofCD45-positive lymphocytes is obtained by washing the bead-cellaggregates three times and can then, dissolved in Trizol®, be passed onfor RNA isolation. CD45 isolates of the MNC fraction before and afterthe screening process are referred to as comparative fraction A and B,respectively.

RNA Isolation

The RNA is extracted and purified from the above cell lines in a mannerknown per se, e.g. with the aid of suitable kits as obtainable fromcommercial suppliers, e.g. from Qiagen and GIBCO-BRL.

mRNA Expression Analysis

The amounts of expressed mRNA are determined by quantitative RT-PCR. ThePCR format is based on the 5′-exonuclease assay known per se (e.g.TaqMan®) and is suitable for use on the TaqMan® 7700 sequence detectorfrom Applied Biosystems (ABI).

The following reagents are employed:

a) Reverse transcription (RT)

-   5× first strand buffer (from Boehringer)-   0.1 M dithiothreitol (DTT)-   RNA guard 38950 U/ml (from Pharmacia)-   random hexamers 500 μg/ml (from Promega)-   dNTPs each 20 mM (from Pharmacia)-   M-MLV 200 U/μl (from Gibco)    b) PCR-   10×TaqMan® buffer (from Perkin Elmer (PE))-   MgCl₂ 25 mM (PE)-   dNTP mix (0.75 μl of each; 2.5 mM)-   ROX solution (100×) (TIB)-   Amplitaq® Gold (PE) (for hot start method)

The reagents mentioned are present in the Perkin Elmer TaqMan® PCR corereagent kit.

To carry out the RT-PCR:

1. Reverse transcription (RT)

Firstly an RT mix composed of 2.35 μl of H₂O, 4 μl of 5× first strandbuffer, 2 μl of 0.1 M DTT, 0.15 μl of RNA guard (38950 U/ml), 0.5 μl ofrandom hexamers (500 μg/ml), 0.5 μl of dNTP mix, 20 mM each, and 0.5 μlof M-MLV (200 U/μl) is prepared.

10 μl of RNA (approx. 1 μg from RNA isolation) are denatured at 70° C.for 1 min, immediately placed on ice and cooled for 3 min, andsubsequently mixed with 10 μl of RT mix free of air bubbles, incubatedinitially at 37° C. for 60 min and then at 95° C. for 3 min, immediatelyplaced on ice and cooled for 3 min.

This reaction mixture comprising the reverse transcript (cDNA) is eithersubjected directly to the PCR or frozen at −20° C.

2. PCR

Firstly a PCR premix composed of 28.5 μl of H₂O, 5.0 μl of buffer (PE),6 μl of ROX solution (100×) (TIB), 6.0 μl of MgCl₂ (25 mM), 3.0 μl ofdNTP mix (0.75 μl of each; 2.5 mM), 1.0 μl of primer (sense; 20pmol/μl), 1.0 μl of primer (antisense; 20 pmol/μl), 0.5 μl of probe (20pmol/μl) and 0.5 μl of Amplitaq Gold (PE 5 U/μl) is prepared.

Special reaction vessels are required for detecting the PCR products onthe TaqMan® 7700 sequence detector. The PE optical tubes in combinationwith the PE optical caps are suitable. 47 μl of PCR premix and 3 μl ofcDNA solution are employed per tube.

A 2-step method with the following thermocycling conditions is thenchosen for the amplification:

-   95° C. 10 min hot start activation-   95° C. 30 sec-   60° C. 60 sec-   20° C. indefinitely number of cycles: 45    2.1 Determination of Manganese Superoxide Dismutase mRNA (MNSOD    mRNA)

The following MNSOD-specific primers and probes are used (MNSOD, SOD2;accession No.: M36693): sense: 5′-GTCACCGAGGAGAAGTACCAGG -3′ (SEQ IDNO:1) antisense: 5′-GGGCTGAGGTTTGTCCAGAA-3′ (SEQ ID NO:2) probe:5′-CGTTGGCCAAGGGAGATGTTACAGCCC-3′ (SEQ ID NO:3)

Size of the PCR product: 131 bp.

2.2 Determination of Thioredoxin Reductase 1 mRNA (TXNRD1 mRNA)

The following TXNRD1-specific primers and probes are used (TXNRD1;accession No.: X91247 cDNA): sense: 5′-GGAGGGCAGACTTCAAAAGCTAC-3′ (SEQID NO:4) antisense: 5′-ACAAAGTCCAGGACCATCACCT-3′ (SEQ ID NO:5) probe:5′-TTGGGCTGCCTCCTTAGCAGCTGCCA-3′ (SEQ ID NO:6)

Size of the PCR product: 158 bp.

2.3 Determination of Glutathione Peroxidase mRNA (GPX1 mRNA)

The following GPX1-specific primers and probes are used (GPX1; accessionNo.: M21304): sense: 5′-CTCGGCTTCCCGTGCAA-3′ (SEQ ID NO:7) antisense:5′-TGAAGTTGGGCTCGAACCC-3′ (SEQ ID NO:8) probe:5′-AGTTTGGGCATCAGGAGAACGCCAAGAA-3′ (SEQ ID NO:9)

Size of the PCR product: 109 bp.

2.4 Determination of Glyceraldehyde-3-Phosphate Dehydrogenase mRNA(GAPDH mRNA)

The following GAPDH-specific primers and probes are used (GAPDH;accession No. X01677): sense: 5′-TGCTGATGCCCCCATGTTC-3′ (SEQ ID NO:10)antisense: 5′-GGCAGTGATGGCATGGACTG-3′ (SEQ ID NO:11) probe:5′-TCAAGATCATCAGCAATGCCTCCTGCA-3′ (SEQ ID NO:12)

Size of the PCR product: 174 bp.

3. Evaluation

For the evaluation, the ratio of cell equivalents of the mRNA to bedetermined to cell equivalents of GAPDH mRNA is found for each of thefractions A or A′ and C, and the ratio of the resulting quotients isfound in turn. Overexpression of the relevant mRNA is present if theratio of the fraction C quotient to the fraction A quotient is more thana limit which is to be defined.

The cell equivalents are based on a cell standard. This cell standard isproduced by mRNA being extracted from a known number of cells (e.g.2×10⁻⁶) of a cell suspension of a carcinoma cell line which expressesthe respective parameter (cell line BT474 for MNSOD, GPX1 and TXNRD1) inthe manner described above, and transcribed into cDNA. This cDNA isincluded in each quantitative analysis in the form of serial dilutions(e.g. 6 dilution levels) and serves as reference system.

Results:

EXAMPLE 1

Healthy donors:

The amounts of MNSOD, TXNRD1 and GPX1 mRNA determined in the isolatedcells (fraction C) for healthy donors (number N) are indicated in table1, specifically as ratio to the amounts of corresponding mRNAs in thecomparative cell fraction (fraction A). TABLE 1 MNSOD, TXNRD1 and GPX1mRNA expression in the blood of healthy donors N Average Standarddeviation Limit MNSOD 9 0.9300 0.2891 1.2 TXNRD1 9 0.9133 0.4166 1.3GPX1 8 3.6888 1.5533 5.2

The cells isolated from blood show a slightly elevated GPX1 expression.The expression of MNSOD and TXNRD1 is unchanged relative to thecomparative cell fraction, that is the lymphocytes.

For the subsequent assessment of the levels of expression measured intumor patients, the levels regarded as positive are those which exceedthe average level plus standard deviation. These levels are indicated aslimit in table 1.

EXAMPLE 2

Tumor Patients

A number of tumor patients with different tumors are included in theinvestigation. The tumors had been diagnosed by various other medicalmethods.

1. Sensitivity

The number of cases among tumor patients (number N) in which theexpression of MNSOD, TXNRD1 and GPX1 mRNA is elevated (positive) in theisolated cells (fraction C) in relation to the cells of the completecell fraction (fraction A′) is indicated below, grouped according totype of tumor. MNSOD Investigations N = 93 Patients N = 90 of whichbreast 41 (40 positive) colon  8 (8 positive) prostate  8 (7 positive)ovary  8 (5 positive) lung  5 (2 positive) bladder  4 (3 positive) liver 2 (1 positive) thyroid  2 (2 positive) others 12 (10 positive) → 78/90= 87% POSITIVE TXNRD1 Investigations N = 93 Patients N = 90 of whichbreast 41 (31 positive) colon  9 (6 positive) prostate  8 (6 positive)ovary  7 (3 positive) lung  5 (1 positive) bladder  4 (3 positive) liver 2 (1 positive) thyroid  2 (1 positive) others 12 (8 positive) → 60/90 =67% POSITIVE GPX1 Investigations N = 89 Patients N = 86 of which breast40 (25 positive) colon  8 (6 positive) prostate  7 (5 positive) ovary  7(3 positive) lung  4 (2 positive) bladder  4 (2 positive) liver  2 (2positive) thyroid  2 (2 positive) others 12 (6 positive) → 53/86 = 62%POSITIVE MNSOD and TXNRD1 and GPX1 Investigations N = 88 Patients N = 850 positive  6/85 = 7% 1 positive  9/85 = 11% 2 positive 33/85 = 39% 3positive 37/85 = 44% → at least 1 gene POSITIVE in 93%

A detection directed at determination of all 3 parameters therefore hasa sensitivity of 93%, while the sensitivity of the individual detectionsis 87, 67 and 62%, respectively.

2. Correlation with One Another

In addition to the sensitivity of each individual parameter, it emergesthat in 74% (58/78) of the cases in which MNSOD expression is elevatedthere is also an observable enhancement of TXNRD1 expression (Pearson'srho correlation coefficient is 0.75). Moreover some, namely 65% (49/75),of the patients with elevated MNSOD expression also have elevated GPX1expression (compare tables 2a, b, c: correlation analysis of TXNRD1 andGPX1 relative to MNSOD and of GPX1 relative to TXNRD1).

The correlation between an elevated MNSOD expression and an elevatedTXNRD1 expression in disseminated cancer cells was not to be expectedbecause the two enzymes have different functions. TABLE 2a Correlationof TXNRD1 relative to MNSOD MNSOD MNSOD negative positive TXNRD1 10/1191% 20/78 26% negative TXNRD1  1/11  9% 58/78 74% positivep = 0.0001 (Pearson's test)

TABLE 2b Correlation of GPX1 relative to MNSOD MNSOD MNSOD negativepositive GPX1 7/11 64% 26/75 35% negative GPX1 4/11 36% 49/75 65%positivep = 0.10 (Pearson's test)

TABLE 2c Correlation of GPX1 relative to TXNRD1 TXNRD1 TXNRD1 negativepositive GPX1 12/27 44% 20/58 34% negative GPX1 15/27 56% 38/58 66%positivep = 0.38 (Pearson's test)3. Correlation with bcl-2 Overexpression

It also emerges that in 100% (5/5) of the cases in which TXNRD1expression is elevated there is an observable enhancement of bcl-2expression (Pearson's rho correlation coefficient is 0.32, and that ofSpearman is 0.58; compare tables 3a, b, c: correlation analysis ofMNSOD, TXNRD1 and GPX1 relative to bcl-2).

This shows that an elevated TXNRD1 expression is involved in theapoptosis blockade associated with elevated bcl-2 expression. TABLE 3aCorrelation of MNSOD relative to bcl-2 bcl-2 bcl-2 negative positiveMNSOD  9/41 22% 0/5  0% negative MNSOD 32/41 78% 5/5 100% positivep = 0.57 (Pearson's test)

TABLE 3b Correlation of TXNRD1 relative to bcl-2 bcl-2 bcl-2 negativepositive TXNRD1 20/41 49% 0/5  0% negative TXNRD1 21/41 51% 5/5 100%positivep = 0.06 (Pearson's test)

TABLE 3c Correlation of GPX1 relative to bcl-2 bcl-2 bcl-2 negativepositive GPX1 10/39 26% 3/5 60% negative GPX1 29/39 74% 2/5 40% positivep = 0.14 (Pearson's test)4. Correlation with Tumor Patients

A comparison between the healthy donors and some of the tumor patients(tables 4a-c) shows that there is a surprisingly clear correlationbetween the measured elevated expression of the MNSOD, TXNRD1 and GPX1genes and the tumor patients. TABLE 4a Comparison between healthy donorsand tumor patients in relation to MNSOD expression Healthy TUMOR N 9 43Average 0.93 4.82 Median 1.01 3.62Wilcoxon p = 0.0004Median p = 0.001

TABLE 4b Comparison between healthy donors and tumor patients inrelation to TXNRD1 expression Healthy TUMOR N 9 44 Average 0.91 2.18Median 0.97 1.72Wilcoxon p = 0.02Median p = 0.01

TABLE 4c Comparison between healthy donors and tumor patients inrelation to GPX1 expression Healthy TUMOR N 8 38 Average 3.69 18.98Median 2.88 13.09Wilcoxon p = 0.0006Median p = 0.0025. Correlation with Recurrences

A further comparison within the group of tumor patients between thosewho have had a recurrence and those who have had no recurrence showsthat there is likewise a surprisingly clear correlation between themeasured elevated expression of the MNSOD, TXNRD1 and GPX1 genes and therecurrences. This applies to all the tumors investigated (tables 5a-c)and in particular to patients with carcinoma of the breast (tables6a-c). A surprising advantage of the method of the invention is that atleast 2, and in particular all 3, parameters correlate better with theclinical course of a cancer than one parameter alone (tables 7a, b).TABLE 5a Comparison between tumor patients without recurrence and thosewith recurrence in relation to MNSOD expression No recurrence RecurrenceN 23 14 Average 3.12 6.17 Median 2.78 5.43Wilcoxon p = 0.02Median p = 0.005

TABLE 5b Comparison between tumor patients without recurrence and thosewith recurrence in relation to TXNRD1 expression No recurrenceRecurrence N 23 14 Average 1.48 2.32 Median 1.17 2.09Wilcoxon p = 0.05Median p = 0.14

TABLE 5c Comparison between tumor patients without recurrence and thosewith recurrence in relation to GPX1 expression No recurrence RecurrenceN 20 14 Average 13.01 23.49 Median 9.20 15.66Wilcoxon p = 0.05Median p = 0.04

TABLE 6a Comparison between patients with carcinoma of the breastwithout recurrence and those with recurrence in relation to MNSODexpression No recurrence Recurrence N 12 4 Average 2.92 8.71 Median 2.736.66Wilcoxon p = 0.03Median p = 0.03

TABLE 6b Comparison between patients with carcinoma of the breastwithout recurrence and those with recurrence in relation to TXNRD1expression No recurrence Recurrence N 12 4 Average 1.53 2.95 Median 1.432.84Wilcoxon p = 0.06Median p = 0.26

TABLE 6c Comparison between patients with carcinoma of the breastwithout recurrence and those with recurrence in relation to GPX1expression No recurrence Recurrence N 11 4 Average 11.12 19.17 Median9.10 15.66Wilcoxon p = 0.13Median p = 0.02

TABLE 7a Comparison between patients with carcinoma of the breastwithout recurrence and those with recurrence in relation to MNSOD,TXNRD1 and GPX1 expression No recurrence Recurrence 0 0/18  0% 0/7  0% 12/18 11% 0/7  0% 2 8/18 44% 3/7 43% 3 8/18 44% 4/7 57%

TABLE 7b Comparison between tumor patients without recurrence and thosewith recurrence in relation to MNSOD, TXNRD1 and GPX1 expression Norecurrence Recurrence 0  1/28  4% 3/29 10% 1  4/28 14% 2/29  7% 2 11/2839% 9/29 31% 3 12/28 43% 15/29  52%6. Correlation with DNA Aberrations

The occurrence of DNA aberrations, i.e. genomic imbalances (G.I.) (cf.Giesing M, et al. Int J Biol Markers, 15-94, 2000) in the isolated cellsalso correlates clearly with elevated expression of the MNSOD, TXNRD1and GPX1 genes, as proved by the results shown in tables 8a-d. Thenumber of genomic imbalances correlates with the number of overexpressedgenes selected from MNSOD, TXNRD1 and GPX1. TABLE 8a Comparison betweenthe absence and the occurrence of genomic imbalances in relation toMNSOD overexpression 1 G.I. ≧2 G.I. SOD > 1.2 16/19 84% 8/8 100%

TABLE 8b Comparison between the absence and the occurrence of genomicimbalances in relation to TXNRD1 overexpression 1 G.I. ≧2 G.I. TXNRD1 >1.3 12/19 63% 5/9 56%

TABLE 8c Comparison between the absence and the occurrence of genomicimbalances in relation to GPX1 overexpression 1 G.I. ≧2 G.I. GPX1 > 5.211/17 65% 8/8 100%

TABLE 8d Comparison between the absence and the occurrence of genomicimbalances in relation to MNSOD, TXNRD1 and GPX1 overexpression 1 G.I.≧2 G.I. 0 0/17 0% 0/7 0% 1 2/17 12% 0/7 0% 2 8/17 53% 2/7 29% 3 8/17 35%5/7 71%mRNA Expression Analysis According to the Invention Using BiochipsmRNA Total Amplification

The RNA amplification takes place as described in Zohlnhöfer D, et al.Circulation 103, 1396-1402, 2001.

Chip Design

Besides various tumor-relevant gene-specific probes, also integrated onthe chip are probes for quantifying MNSOD, GPX2, GPX3 and TXNRDexpression.

The following MNSOD-, GPX2-, GPX3- and TXNRD-specific probes are used:MNSOD: (SEQ ID NO:19) GAACAACAGGCCTTATTCCACTGCTGGGGATTGATGTGTGGGAGCACGCTTACTACCTTC TXNRD1: (SEQ ID NO:20)CGTGTTGTGGGCTTTCACGTACTGGGTCCAAATGCTGGAGAAGTTACACA AGGCTTTGCA GPX2: (SEQID NO:21) TACAGCCGCACCTTCCCAACCATCAACATTGAGCCTGACATCAAGCGCCT CCTTAAAGTTGPX3: (SEQ ID NO:22) CTCTTCTGGGAACCCATGAAGGTTCACGACATCCGCTGGAACTTTGAGAAGTTCCTGGTGProduction of the Oligonucleotide ArraysProduction of the Probesa) oligonucleotides (60-mers)

-   The oligonucleotides are produced in a manner known per se by    solid-phase synthesis with the phosphoramidite method.    Oligonucleotides which are coupled via the 3′-OH to the solid phase    and have DMTr-protected 5′-OH and the above sequence are    synthesized.    b) Quinone-spacer construct-   This is synthesized in a manner known per se from    anthraquinone-2-carboxylic acid, mono-Boc-1, 3-propane-diamine and    hexaethylene glycol.    c) Quinone-spacer-oligonucleotide construct-   After assembly of the above sequences, the DMTr protective group at    the 5′ end is again removed under acidic conditions with ZnBr₂, and    the free 5′-OH is reacted with the    2-cyanoethyl-N,N-diisopropylchloro-phosphoramidite-activated    quinone-spacer construct.

The quinone derivatives synthesized in this way therefore have astructure of the general formula AQ-CO—NH—(CH₂)₃—NH—(CH₂)2—(OCH₂CH₂)₅—O—PO₂-5′ (SEQ ID NO:19-22)-3′.

The other cancer-relevant gene-specific probes are also produced in ananalogous manner.

Coupling of the Probes to the Array Surface

An aqueous solution (2 mM calcium chloride; 1% by vol. 1-propanol) ofeach desired quinone derivative (10 μM) is applied using apiezodispenser (1-channel; Genesis NPS 100/4 with Active Tip M, TECANAG, Hombrechtikon, CH) in a 400 μm grid on polycycloolefin (Zeonex 480R;Zeon). The drop size is about 0.5 nl. After the spots have dried, thesupport is irradiated with UV light for 1 min. The support is thenwashed and dried in air at room temperature. The diameters of theresulting spots are about 120 to 180 μm.

Hybridization

The mixtures obtained from the RNA amplification are diluted in asuitable manner with 2×SSPE buffer and pipetted onto the array. It isincubated at 60° C. for 16 hours. After two washing steps with 1×SSPEbuffer at 60° C., Cy5-streptavidin (Amersham Pharmacia Biotec) is addedand incubated at room temperature for 15 min.

Evaluation of the Array

The fluorescence is determined using a CCD camera scanner.

Example 3

Tumor Patients

FIG. 1 shows a CCD contact exposure image of the fluorescence radiationemitted from the array after hybridization with cDNA single-strandedfragments which were generated in the manner described above by means ofmRNA total amplification from the tumor cell fraction C and thecomparative cell fraction A′.

Overexpression of MNSOD and GPX2 in the tumor cell fraction is clearlyevident. The measurement underlines the increased sensitivity andspecificity of the detection method of the invention compared with othertumor cell-detecting genes.

1. A method for investigating a body fluid for cancer cells, where theexpression of at least 2 genes which are selected from the groupconsisting of I) manganese superoxide dismutase genes; ii) thioredoxinreductase genes; and iii) glutathione peroxidase genes is determined onat least one cell-containing fraction of the body fluid:
 2. The methodas claimed in claim 1, wherein the expression of at least one manganesesuperoxide dismutase gene, of at least one thioredoxin reductase geneand of at least one glutathione peroxidase gene is determined.
 3. Themethod as claimed in claim 1 wherein the body fluid is selected fromblood, bone marrow, lymph, sputum, ravages, puncture fluids, ascites,mucosal smears, exudates, urine and stool.
 4. The method as claimed inclaim 1 wherein the cell-containing fraction is obtained from the bodyfluid with enrichment of cancer cells.
 5. The method as claimed in claim1 wherein the cell-containing fraction is obtained from the body fluidwith enrichment of cancer cells, and the expression of the genes in thecell-containing fraction is determined, a further cell-containingfraction of the body fluid or of a comparable biological sample isprovided, and the expression of the genes in the further cell-containingfraction is determined, and the expression for each gene in thecell-containing fraction is compared with its expression in the furthercell-containing fraction.
 6. The method as claimed in claim 5, whereinthe comparable biological sample is derived from the individual whosebody fluid is investigated for cancer cells.
 7. The method as claimed inclaim 6 wherein it is determined whether expression of the genes in thecell-containing fraction is elevated by comparison with the expressionof the genes in the further cell-containing fraction.
 8. (canceled) 9.An analysis kit comprising I) means for determining the expression of atleast one manganese superoxide dismutase gene; ii) means for determiningthe expression of at least one thioredoxin reductase gene; and iii)means for determining the expression of at least one glutathioneperoxidase gene, and optionally further usual means for carrying out themethod as claimed in claim
 1. 10. An analysis kit as claimed in claim 9,comprising I) sequence-specific primers and/or probes for determiningthe expression of at least one manganese superoxide dismutase gene; ii)sequence-specific primers and/or probes for determining the expressionof at least one thioredoxin reductase gene; and iii) sequence-specificprimers and/or probes for determining the expression of at least oneglutathione peroxidase gene.
 11. The method as claimed in claim 1,wherein the expression of at least one manganese superoxide dismutasegene and of at least one further gene selected from the group consistingof thioredoxin reductase genes and glutathione peroxidase genes isdetermined.
 12. The method as claimed in claim 1, which is foridentifying disseminated cancer cells in the body fluid.
 13. The methodas claimed in claim 12, wherein the elevated expression of at least oneof said genes indicates the presence of disseminated cancer cells in thebody fluid.
 14. The method as claimed in claim 1, which is for earlydiagnosis of a tumor.
 15. The method as claimed in claim 14, wherein theelevated expression of at least one of said genes indicates the presenceof a tumor.
 16. The method as claimed in claim 1, which is forestimating the risk to develop a metastasis or a recurrence.
 17. Themethod as claimed in claim 16, wherein the elevated expression of atleast one of said genes indicates a risk to develop a metastasis or arecurrence.